Are Bcr-Abl KD mutations already detectable at the time ... - siesonline

LAL:significato clinico e biologicodelle mutazioni di Bcr-AblSimona SoveriniDipartimento di Ematologia e Scienze Oncologiche “L. e A. Seràgnoli”Università di Bologna

The vast majority of Ph+ ALL patientstreated with imatinib relapse withevidence of a Bcr-Abl KD mutation

Imatinib-resistant pts with Ph+ ALL have thehighest incidence of Bcr-Abl KD mutationsData of the GIMEMA WP on CML on 655 IM-resistant ptsPhase at the time of IM failure:CP (IM first-line) AP/BC CML Ph+ ALL30%64%76%resistant pts harbouring Abl KD mutations

T315I is the single most frequent Bcr-AblKD mutation in imatinib-resistant Ph+ ALL66 mutations in 62/82 ptsresistant to imatinibT315I mutations: 17/66 (26%)*P-loop mutations: 26/66 (39%)P-loopcatalyticdomainactivationloopM244VL248VG250EQ252R/HY253F/HD276GT277AE255K/VE279KV289AE281A/KE292VM343TH396R/PL364IM351TL387M/FL384MF359V/IS417Y/FT315I E355G/DF382L E453G/KV379IF311L/I F317LE459K/QA380TF486S* Frequency of T315I mutations in imatinib-resistant CML: ~10-12%

Second-line TKIs in imatinib resistant Ph+ ALLpatients have limited efficacyCHRmCgRF311IV299LNELG250ECHRCCgRF317LY253HCHRCCgRT315IWTCHR100T315IE255KCHRCCgRE255K,T315ID276GCHR PCgR100T315IWTCHR100F317LE255KCHRCCgRT315IY253HCHRCCgRT315A, F317LM351TCHRCCgR90M351T, F317LWTCHRF317IL387ML387M,F317Lmonths on dasatinib0 1 2 3 6 9 12 15

Inhibiting Bcr-Abl in Ph+ ALL is likeshooting at a moving targetdasatinibimatinib 800nilotinib0141618monthsMale, 18 yrs, Ph+ ALLWTT315AY253HY253HT315AWTT315AWTT315AT315AT315AT315AY253HWTT315AG250ET315AG250ET315AG250ET315AY253HWT3 WTWT CCgR, MMRWTWTrelapserelapserelapseimatinibdasatinibnilotinib0121822monthsMale, 69 yrs, Ph+ ALLG250EG250EWTG250EWTWTWTWTG250EF317LG250E WTF317LG250EF317LY253HY253HWT G250EF317L WTG250EF317L Y253Hrelapserelapserelapse

In ALL genetic instability is high and allowsthe Ph+ clone to rapidly escape inhibitionNovel TKIs are active against the vast majority of IM-resistant mutations, butthey inexorably retain one or some insensitive mutationsDisease burdenIMATINIB INHIBITOR 2 INHIBITOR 3 ??timeRELAPSE 1 RELAPSE 2 RELAPSE 3BCR-ABL KD sequenceWT(?)IM-res mutationAccumulation of compounddrug-resistant mutations

Are Bcr-Abl KD mutations alreadydetectable at the time of diagnosis?• In Ph+ ALL pts treated with imatinib, resistance-associatedBcr-Abl KD mutations arise more frequently and more rapidlythan in CML pts• Imatinib does not to induce mutations, but simply selectspre-existent mutated clonesIn how many newly diagnosed Ph+ ALL can we detect Bcr-AblKD mutations at low levels?

Are Bcr-Abl KD mutations alreadydetectable at the time of diagnosis?• German experience: Bcr-Abl KD mutations detected by D-HPLC at the time of diagnosis in:- 9/22 (41%) cases in elderly pts (Pfeifer et al, Blood 2007)– 6/34 (18%) cases in younger pts (Pfeifer et al, EHA 2008)• Lower detection limit of D-HPLC is routinely 5-10%• What happens if we increase the sensitivity of ourscreening method?

Patients (I)→ RNA samples collected at the time of diagnosis from13 patients enrolled on a phase II study of the treatmentof adult de novo Ph+ ALL with dasatinib (GIMEMA LAL1205)DASATINIB 70 mg BIDPDNi.t. mtxi.t. mtx-6 0 +1 +22 +32 +43+84 days

Patients (II)NSexAgeBcr-Abl typeStatus (time fromthe start of dasatinib,mo)Mutation atdiagnosis(direct seq)Mutation atrelapse(direct seq)1M49p190Relapsed (10)WTT315I2M56p210Relapsed (6)WTT315I3M56p210Relapsed (7)WTWT4F47p190Relapsed (4)WTWT5F74p210Relapsed (8)WTT315I6F58p210Relapsed (7)WTE255K7F59p210Relapsed (2)WTT315I8F30p210Relapsed (6)WTT315I9F57p190Remission (13)WTN.A.10F34p190Remission (5)WTN.A.11M52p190Remission (12)WTN.A.12F73p210Remission (23)WTN.A.13M72p190Remission (20)WTN.A.N.A., not applicable

Experimental designWT Y342Y E292GBCRKDABL1.Amplification ofthe BCR-ABL KD(a.a 206-524)by nestedRT-PCR andligation into aplasmidlacZlacZpCR2.1TOPOvectorAMP R4.Directsequencing of150 to 200independentclones2.Transformationof E. colicompetent cells3.Plating andovernightincubationat 37°C

Results (I)Aberrant Bcr-Abl KD sequences were detectedin ALL the patientsinsertionsdeletionspoint mutationsmissense (→ amino acid change)silent (→ no amino acid change)nonsense (→ premature stop codon)N216139184

Results (II)Point mutations were randomly and uniformly distributedall over the KDMost of them were not likely to be clinically relevantP-loop C-helix SH3 contact SH2 contact A-loopP216P K245R Y257H E275E E292G T315I E334E I360T T392I Q477stopK222E G249S Y264C E281K I293T F317L V335V A366A H396PD223N G250RA288T V304I/A T319A N336D R367stop I403TG227G Q252stop V289A R307Q Y326Y A337A K378E W405CV228A Y253YL327L Y342Y A380A A407AS229T/P G254EA344T L384PC346stop T389TE352G/KY353YMutations that have been reported in imatinib-resistant pts are highlighted

N12345678910111213Status (time fromthe start ofdasatinib, mo)Relapsed (10)Relapsed (6)Relapsed (7)Relapsed (4)Relapsed (8)Relapsed (7)Relapsed (2)Relapsed (6)Remission (13)Remission (5)Remission (12)Remission (23)Remission (20)Results (III)Each In In two contrast, patients, had an the F317L evidence T315I detected observed of two at to diagnosis at twelve relapse different failed could to already mutations expandbedetected at the time of diagnosisSexMMMFFFFFFFMFMAge49565647745859305734527372Bcr-Abltypep190p210p210p190p210p210p210p210p190p190p190p210p190Mutations* at diagnosis (total no. of clones sequenced)L266P; A337A (150)K222E; V289A; V335V; K378E (180)V228A; R307Q; W405C (150)E275E; E281K; T319A; N336D (200)K245R; I293T; V304I; D363D; R367stop; K378E; 39 bp ins (180)G250R; L327L; I360T (150)G227G; I293T; V304I; T315I; C346stop; W405C (200)P216P; Y264C; T315I; A344T (150)D223N; S229T; Q252stop; L384P; T392I; A407A;del(cggga)1343 (200)S229P; K245R; A288T; 171 bp ins (200)G254E; Y326Y; E334E (150)E286E; E352K; A380A; T389T; Q477stop (150)G249S; Y253Y; Y257H; E292G; T304A; F317L; Y342Y; E352G;Y353Y; A366A; H396P; I403T (200)* Each mutation was detected by bidirectional sequencing in 2 to 5 independent clonesMutation atrelapse(direct seq)T315IT315IWTWTT315IE255KT315IT315IN.A.N.A.N.A.N.A.N.A.

Results (IV)55/61(90%) nucleotide substitutions were transitionsTransitions:G>AA>GT>CC>TTotalTransversions:G>TA>TT>AT>GA>CTotal1514131355211116Such a high prevalenceof transitions (whichnormally occur 1.4 timesmore frequently thantransversions) suggeststhat a specific mechanismgenerating mutations ispredominant

Take-home messagesa) Bcr-Abl KD mutations can probably be found at thetime of diagnosis in all Ph+ ALL ptsb) TKI-resistant mutations present at low levels atdiagnosis do not always outgrow and lead to relapse,probably because some of them arise in cell cloneswith limited self-renewal capacityc) It is essential to understand the molecularmechanism(s) sustaining this “mutator phenotype” aswell as how to therapeutically interfere with them

AcknowledgmentsDepartment of Hematology and Oncological Sciences “L. e A. Seràgnoli”,University of Bologna, ItalyMichele BaccaraniGiovanni MartinelliAlessandra GnaniSabrina ColarossiStefania PaoliniIlaria IacobucciCristina PapayannidisAnnalisa LonettiMarilina AmabileDepartment of Hematology and Cellular Biotechnologies, “La Sapienza” University,Rome, ItalyRobin FoàAntonella VitaleMarco VignettiLoredana EliaGiovanna MeloniAnna GuariniFranco MandelliGIMEMA ALL WORKING PARTY